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Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD...

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Autores principales: Bardelang, Philip, Murray, Ewan J., Blower, Isobel, Zandomeneghi, Sara, Goode, Alice, Hussain, Rohanah, Kumari, Divya, Siligardi, Giuliano, Inoue, Katsuaki, Luckett, Jeni, Doutch, James, Emsley, Jonas, Chan, Weng C., Hill, Philip, Williams, Paul, Bonev, Boyan B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10196373/
https://www.ncbi.nlm.nih.gov/pubmed/37214482
http://dx.doi.org/10.3389/fchem.2023.1113885
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author Bardelang, Philip
Murray, Ewan J.
Blower, Isobel
Zandomeneghi, Sara
Goode, Alice
Hussain, Rohanah
Kumari, Divya
Siligardi, Giuliano
Inoue, Katsuaki
Luckett, Jeni
Doutch, James
Emsley, Jonas
Chan, Weng C.
Hill, Philip
Williams, Paul
Bonev, Boyan B.
author_facet Bardelang, Philip
Murray, Ewan J.
Blower, Isobel
Zandomeneghi, Sara
Goode, Alice
Hussain, Rohanah
Kumari, Divya
Siligardi, Giuliano
Inoue, Katsuaki
Luckett, Jeni
Doutch, James
Emsley, Jonas
Chan, Weng C.
Hill, Philip
Williams, Paul
Bonev, Boyan B.
author_sort Bardelang, Philip
collection PubMed
description Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.
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spelling pubmed-101963732023-05-20 Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate Bardelang, Philip Murray, Ewan J. Blower, Isobel Zandomeneghi, Sara Goode, Alice Hussain, Rohanah Kumari, Divya Siligardi, Giuliano Inoue, Katsuaki Luckett, Jeni Doutch, James Emsley, Jonas Chan, Weng C. Hill, Philip Williams, Paul Bonev, Boyan B. Front Chem Chemistry Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo. Frontiers Media S.A. 2023-05-05 /pmc/articles/PMC10196373/ /pubmed/37214482 http://dx.doi.org/10.3389/fchem.2023.1113885 Text en Copyright © 2023 Bardelang, Murray, Blower, Zandomeneghi, Goode, Hussain, Kumari, Siligardi, Inoue, Luckett, Doutch, Emsley, Chan, Hill, Williams and Bonev. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
Bardelang, Philip
Murray, Ewan J.
Blower, Isobel
Zandomeneghi, Sara
Goode, Alice
Hussain, Rohanah
Kumari, Divya
Siligardi, Giuliano
Inoue, Katsuaki
Luckett, Jeni
Doutch, James
Emsley, Jonas
Chan, Weng C.
Hill, Philip
Williams, Paul
Bonev, Boyan B.
Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title_full Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title_fullStr Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title_full_unstemmed Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title_short Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate
title_sort conformational analysis and interaction of the staphylococcus aureus transmembrane peptidase agrb with its agrd propeptide substrate
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10196373/
https://www.ncbi.nlm.nih.gov/pubmed/37214482
http://dx.doi.org/10.3389/fchem.2023.1113885
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